Method for delivering a bioactive molecule to a cellular target

ABSTRACT

Hybrid reagents comprising a first portion having an affinity for a cellular target and a second portion having an affinity for a bioactive molecule are described, said hybrid reagents being capable of selectively releasing the bioactive molecule in response to a change in pH. The hybrid reagents of the present invention can be used diagnostically or therapeutically.

GOVERNMENT FUNDING

The invention described herein was supported in whole or in part by theNational Institutes of Health. The United States Government has certainrights to this invention.

The application is a division of application Ser. No. 07/998,754 filedDec. 28, 1992, now U.S. Pat. No. 5,501,854 which is a continuation ofapplication Ser. No. 07/482,001 filed Feb. 16, 1990 now abandoned.

BACKGROUND OF THE INVENTION

Hybrid antibodies are antibodies or aggregates of antibodies which arespecific for two different antigens. Hybrid antibodies can comprise asingle antibody or fragment having a bispecific antigen binding region(two different variable regions) or aggregates of two or more antibodiesof different specificities.

Different methods of preparing hybrid antibodies have been reported.Auditore-Hargreaves teaches processes for preparing hybrid antibodies bygenerating "half molecules" from two parent antibodies and subsequentlyassociating different half molecules. See U.S. Pat. Nos. 4,470,925(1984) and 4,479,895 (1984). Using this process, various hybridantibodies were prepared with specificities for horseradish peroxidase,glucose oxidase and theophylline.

Reading describes production of antibodies having binding specificitiesfor two desired antigens using a quadroma cell or a trioma cell. SeeU.S. Pat. No. 4,474,893 (1984). The quadroma cell is the fusion productof two different hybridoma cells, each of which produce an antibody witha different specificity. A trioma cell is the fusion product of ahybridoma and a lymphocyte which produces antibodies with two differentbinding specificities.

Segal et al. describe target specific crosslinked heteroantibodies whichare used as cytotoxic agents in U.S. Pat. No. 4,676,980 (1987). Staerzet al. (1986), PNAS, 83:1453-1457, teach the use of a hybrid antibodythat can focus effective T cell activity and Milstein et al. (1983),Nature, 305:537-539, describe the use of hybrid antibodies inimmunohistochemistry.

Raso et al., Cancer Research, 41:2073-2078 (1981) disclose the use ofhybrid antibodies with dual specificity for the plant toxin, ricin, andimmunoglobulin-bearing target cells. The hybrid antibodies wereconstructed in vitro and the attachment of the hybrid antibody-ricincomplex to the human target cells was observed using fluorescein labeledantibodies. Upon binding, the human target cells were selectively killedby the hybrid-delivered toxin.

Prior to the use of hybrid antibodies, chemical crosslinking ornonspecific absorption methods were used to couple drugs and/or toxinsto antibody carriers. These agents possess certain limitations due tothe nature of the linkage. The linkage may alter the drug or toxin suchthat the therapeutic or toxic activity is reduced. Moreover, cleavage ofthe covalent bond may be rate-limiting for the action of toxin insidethe cell.

The use of hybrid antibodies obviated some of the problems encounteredwith chemical crosslinking or non-specific absorption methods; however,new problems were created. Because the drug or toxin is bound to anantibody, the therapeutic or toxic activity is generally inhibited.Hybrid antibody-delivered toxins or drugs are inactive when bound to theantibody and only become active upon release. However, the hybridantibodies currently available have no mechanism for releasing the toxinor drug from the respective antibody binding region when the hybridantibody reaches the target site or the interior of the cell. Instead,they rely on fortuitous dissociation. As a result, relatively largequantities of hybrid antibodies containing drugs or toxins must beadministered, because only a small amount of the drug or toxin willdissociate and become active.

SUMMARY OF THE INVENTION

This invention pertains to hybrid reagents comprising a first portionhaving an affinity for a cellular target (e.g., antibody, virus, ligand,receptor or molecule) and a second portion having an affinity for abioactive molecule (e.g., a toxin, drug, enzyme or metal). The hybridreagents can be administered in vivo where the bind to the externalsurface of a cell. Once bound to the cell, receptor-mediated endocytosisserves to pinch off the surface of the cell forming an endosome, whichhas a lower pH than either outside or within the rest of the cell. Inresponse to the pH change inside the endosome, the hybrid reagents ofthe present invention selectively release the bioactive molecule. Oncereleased, the bioactive molecule is free to perform its function.

Therefore, a major advantage of hybrid reagents of this invention overcurrently available hybrid antibodies, which rely on fortuitousdissociation of bioactive molecules, is that less of the hybrid andbioactive molecule need to be administered to produce the desireddiagnostic or therapeutic effect.

The present invention also encompasses pharmaceutical compositionscomprising said hybrid reagents having a bioactive molecule boundthereto, methods of immunotherapy and a method for selecting antibodiesor fragments thereof capable of binding a bioactive molecule at one pHand releasing that molecule in response to a change in pH.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram depicting the delivery to a cell of abioactive molecule from a hybrid reagent by receptor mediatedendocytosis and release of the bioactive molecule in response to thelower pH found within a cellular endosome.

FIG. 2 is a graph plotting the percent dissociation (i.e., release) ofmonoclonal antibody 6B3 from diptheria toxin over 100 minutes time at apH of 4.5 and temperatures of 22° C. and 37° C.

FIG. 3 is a graph plotting the percent dissociation of monoclonalantibody 6B3 from diptheria toxin over 30 minutes time at pH 5.0 and pH4.5 at 37° C.

FIG. 4 is a graph plotting the percent dissociation of monoclonalantibodies 5A7 and 1F3 from diphtheria toxin over 60 minutes time at pH5.0 at 37° C.

FIG. 5 is a graph plotting the percent incorporation of ³ H leucine over180 minutes time as a measure of protein synthesis inactivation bynative diphtheria toxin and hybrid-delivered CRM107 in H-meso cells.

FIG. 6 is a graph plotting the toxicity dose-response curve for hybridsand conjugates incubated for 16 hrs. with transferrin receptor positiveCEM cells.

FIG. 7 is a graph plotting the toxicity dose-response curve for HIV andtransferrin receptor directed hybrids on HIV-infected 8E5 cells.

DETAILED DESCRIPTION OF THE INVENTION

The hybrid reagents of this invention comprise a first portion having anaffinity for a cellular target and a second portion having an affinityfor a bioactive molecule (e.g., a toxin, drug, metal or an enzyme). Thehybrid reagents can be administered in vivo where they bind to theexternal surface of a cell. Once bound to the cell, receptor-mediatedendocytosis serves to pinch off the surface of the cell forming anendosome, Which has a lower pH than either outside or within the rest ofthe cell. In response to the change in pH within the endosome, thehybrid reagents selectively release the bioactive molecule. The firstportion of the hybrid can be, for example, a ligand (e.g., transportproteins such as transferrin, interleukin-2, LDL), a growth factor(e.g., EGF, PDGF), an antibody, a hormone, a receptor molecule (e.g.,recombinant CD4), a virus, or a fragment thereof and the second portionis an antibody or an antibody fragment.

The first portion of the hybrid reagent has an affinity-for a cellulartarget, such as an antigenic or receptor site on the surface or inside acell (i.e., a cell surface antigen or cell surface receptor). Examplesof cellular targets are Ig, common acute lymphoblastic leukemia antigen(CALLA), B1, gp26, Ia, transferrin receptor, EBV transformation antigenand the receptors for ligands such as interleukin-2, MSH, insulin,thyroglobulin, LHRH and NGF. Viral proteins on the surface of infectedcells (e.g., HIV-infected T-lymphocyte) can also serve as targets forantibody and receptor guided hybrid reagents.

The second portion of the hybrid reagent is an antibody or antibodyfragment chat has an affinity for a bioactive molecule at one pH andreleases the bioactive molecule in response to a change in pH. Thisbonding and release may be due to a number of mechanisms. For example,the second portion of the hybrid reagent may have an affinity for abioactive molecule that undergoes a conformational change in response toa change in pH. Such molecules can be identified by using physical orother methods known in the art (e.g., circular dichroism, fluorescence).As another example, the second portion of the hybrid reagent mayionically bond to a bioactive molecule at one pH and the ionic bond maybreak in response to a change in pH.

A method for isolating antibodies that dissociate from molecules inresponse to a change in pH is described in detail in Example 1. Ingeneral, antibodies against a bioactive molecule are prepared usingknown techniques. Clone supernatants are then assayed for the ability tobind the molecule at the first selected pH. Clones testing positive forbinding ability are screened to isolate those that release the moleculeat a second selected pH. For example, antibodies that bind a bioactivemolecule at physiologic pH (pH about 6.5 to 7.5) can be tested toisolate those clones that release the molecules at acidic pH (pH lessthan 6.5).

Examples of bioactive molecules are plant or bacterial toxins, drugs,enzymes and metals. Examples of useful toxins are diphtheria toxin,pseudomonas exotoxin, ricin, pokeweed antiviral peptide (PAP), andtricathecum. The toxins can also be genetically or chemically altered ormutated such as CRM107 (Laird J. Virol., 19:220-227 (1976)) and HA48DTand HA51DT (Myers et al., J. Biol. Chem., 263:17122-17127 (1988)). Drugswhich can be used in the invention are for example, interferon, insulin,and methotrexate. Examples of metals which can be used in the inventionare radiometals (e.g., Tc-99m, In-111, Cu-67, Pd-109, Pd-103, Re-188,Au-198, Au-199, Ru-97, Hg-197, Ag-111, Bi-212, Os-191 and Pb-203) andnon-radioactive metals (e.g., zinc).

FIG. 1 illustrates receptor-mediated endocytosis of a hybridreagent-molecule complex. The first portion of the hybrid reagent bindsto the external surface of the cell, which becomes pinched off to forman endosome. Endosomes have a pH lower than (e.g., pH about 4.5-5.5) thepH either outside or within the rest of the cell (e.g., pH about6.5-7.5) (Geisow, M. L. and W. H. Evans, Ext. Cell Res., 150:36-46(1984)). Therefore, by using a hybrid reagent in which the first portionhas an affinity for a cell surface component and the second portion hasan affinity for a bioactive molecule at physiologic pH and dissociatesfrom the bioactive molecule in response to acidic pH, a molecule can bedelivered into a cell and released within acidic compartments of cells,such as cell endosomes.

The hybrid reagents can be produced by joining together the first andsecond portions using known techniques (e.g., chemical coupling, cellfusion, or genetic engineering techniques). The hybrid reagents arepreferably made by chemically coupling the two portions together. Forexample, a disulfide linkage using N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) as the crosslinking agent can be used (Raso et al.,NATO Advanced Studies Institute, 82:119-138 (1984)). Both portionsbecome sparingly substituted with pyridyldisulfide groups which arereduced to thiols on one of the portions. Upon mixing of the twoportions, the free thiols on one of the portions readily reacts with theunreduced groups on the second portion and form disulfide linkages. Theresulting hybrids can then be purified using gel filtration.

When the first and second portions of the hybrid reagent are bothantibodies, two whole parental antibodies may be joined together toproduce the hybrid reagent (i.e., hybrid antibody). A variety ofcrosslinking agents, such as protein A, carbodiimide, andN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) can be used to linkthe whole parental antibodies (Kranz et al., Proc. Natl. Acad. Sci. USA,78:587 (1981); U.S. Pat. No. 4,474,893)).

The hybrid antibodies can also be produced by chemically joiningparental antibody fragments containing a sufficient portion of theantigen binding region to allow the fragment to bind to its respectiveantigen (Nisonoff et al., Arch. Biochem. Biophys., 93:460-467 (1961) andRaso et al., Cancer Research, 41:2073-2078 (1981)). The two types ofparental antibodies (i.e., one type will become the first portion of thehybrid antibody and the other type will become the second portion) canthen be separately digested with pepsin. Bivalent F(ab')₂ molecules areobtained after a separation step such as chromatography. Equal amountsof the 2F(ab')₂ types can then be mixed and after reducing theirinter-heavy chain disulfide linkages, the resulting Fab' fragments areallowed to randomly reassemble into F(ab')₂ dimers with dualspecificity. The dual specificities of the hybrid product can beverified using cell-based and solid phase assays which use radioactiveor fluorescent probes (Raso, V., Immunol. Reviews, 62:93-117 (1982)).

Alternatively, the intrinsic disulfide links of the F(ab')₂ moleculescan be reduced to thiois and the vicinal thiols generated can bestabilized (e.g., with sodium arsenite). Ellman's reagent can be used toactivate the vicinal thiols on one type of the Fab' fragments. Uponmixture of the reduced Fab' fragment with an activated Fab' fragment, anexclusively bi-specific hybrid will be formed (Brennan, M., et al.,Science, 228:81-83 (1985)).

The hybrid antibodies can also be produced using cell fusion techniquesas described in U.S. Pat. No. 4,474,893, to Reading. In this technique,hybridoma cells which secrete the parental antibodies are fused togetherto form quadroma or trioma cells. These quadroma and trioma cellssecrete bi-specific antibodies possessing the antigen binding regions ofboth parental antibodies.

In addition, the hybrid antibodies can be produced using geneticengineering techniques. In these procedures, DNA encoding the heavy andlight chain variable regions of each of the parental antibodies areintroduced into an appropriate host cell, preferably a lymphoid cell(e.g., a myeloma cell). The transformed cell can then synthesize,assemble and secrete the hybrid antibody.

The parental antibodies used to produce the hybrid antibody can beselected from those presently available or can be specially prepared.The parental antibodies can be obtained using conventional monoclonalantibody methodology, (e.g., the standard somatic cell hybridizationtechniques of Kohler and Milstein, Nature, 256:495 (1975)).

Suitable antibodies which are specific towards tumor associated antigensand are therefore appropriate to comprise the first portion of thehybrid reagent, are for example, 7D3, directed against the humantransferrin receptor, (Griffin et al., Cancer Res., 47:4266 (1987));C19, directed against the carcinoembryonic antigen, (Griffin et al., J.Biol Resp. Modif., 1:194 (1982)); 260F9, directed against a breastcancer antigen, (Bjorn et al., Cancer Res., 45:1214 (1985)); 96.5directed against a melanoma associated antigen, (Casellas et al., In J.Cancer, 30:437 (1982)); 45-2D9, directed against an oncogene product,(Roth et al., J. Immunol., 136:2305 (1986)) and J-5, directed againstthe common acute lyphoblastic leukemia antigen, (Raso et al., CancerRes., 42:457 (1982)).

Suitable antibodies which are specific towards diptheria toxin and arecapable of releasing the toxin in response to a change in pH fromphysiologic to acidic, are D5E8, D1F3, D3E1, D6B3, D5D5, D1D5, D5F5 andD4B7. These antibodies are therefore appropriate to comprise the secondportion of the hybrid reagent.

The hybrid reagents described herein can be used diagnostically. Forexample, hybrid molecules comprising a first portion which has anaffinity for a tumor cell and a second portion which has an affinity fora radiometal can be used to deposit radiometal within tumor cells andthereby obtain a scintographic image of the tumor.

Hybrid reagents of this invention can also be used therapeutically. Forexample, hybrid molecules comprising a first portion having an affinityfor a viral-associated antigen (e.g., an HIV antigen) or aviral-associated receptor and a second portion having an affinity for abioactive molecule, can be used therapeutically to kill or otherwisemodify virus infected cells. Similarly, hybrid molecules comprising afirst portion having an affinity for a tumor-associated antigen or atumor-associated receptor and a second portion having an affinity for abioactive molecule can be used therapeutically to kill or otherwisemodify tumor cells.

When the hybrid reagent described herein is used in a pharmaceuticalcomposition, it can be administered by a wide variety of techniques. Forexample, intravenously, parenterally, transdermally subcutaneously orvia an implanted reservoir containing the hybrid molecule. The form inwhich the hybrid molecule will be administered (e.g., solution,emulsion) will depend on the route by which it is administered. Thequantity of the hybrid molecule to be administered will be determined onan individual basis and will be based at least in part on considerationof the individual's size, the severity of the symptoms to be treated,and the result sought.

This invention is further illustrated by the following examples.

EXAMPLE 1 The Isolation of Anti-Diphtheria Toxin Antibodies Capable ofReleasing a Molecule at a Selected pH

Mice were immunized with progressively increasing doses of activediphtheria toxin (1 μg-3 μg I.P.) or a high dose of formalin-inactivateddiphtheria toxoid (100 μg I.P.). Following a booster injection of theimmunogen, spleens were removed and fused with NS-1 cells to generatehybridomas (Kohler and Milstein, Nature, 256:495 (1975)). Supernatantsfrom microtiter wells with clones were assayed for the ability to bind¹²⁵ I-diphtheria toxin using a polyethylene glycol precipitation method.Antibody positive supernatants usually bound 25,000 cpm while negativesand controls bound only 4,000 cpm. In a typical fusion approximately 35positive clones were obtained from the spleen of a single animal.

A second assay was developed in order to examine the influence of pH onthe interaction between diphtheria toxin and the different monoclonalantibodies. Diphtheria toxin (100 μl at 300 μg/ml) was absorbed topolyvinyl microtiter wells, excell was washed off with PBS. Antibody(100 μl at 1-50 μg/ml) was then added, allowed to react for two hoursand the plate was washed with PBS. Attached antibody was revealed bysubsequent addition of a ¹²⁵ I-goat antimouse IgG reagent (backgroundwas approximately 100 cpm, positive clones bound approximately1,000-3,000 cpm).

To test for pH effects on toxin release, the antibody was allowed tobind to the immobilized diphtheria toxin for two hours in replicatewells and then a small volume of concentrated buffer was added toprovide a final pH of 7.0, 5.0 or 4.5. Dissociation was allowed toproceed for different time intervals (5-90 minutes) at either 23° C. or37° C. (normal body temperature). Released antibody was quickly washedoff the plates with PBS and the amount remaining was quantified using a¹²⁵ I-goat antimouse IgG probe. This method was used to identify 23clones producing antibody which rapidly dissociated from diphtheriatoxin at a pH of 4.5 and eight clones having antibody that was sensitiveto release at a pH of 5.0. No release occurred at a pH of 7.0.

The time-course of dissociation at pH 4.5 for one of these monoclonalantibodies (D6B3) is shown in FIG. 2. At 23° C. the rate of release wasslower and less complete than at 37° C. Approximately 80 percent of theantibody initially bound dissociated from diphtheria toxin and most ofthis occurred within the first 5 minutes. It is known that thediphtheria toxin remains attached to the assay plate since binding ofmonoclonal antibodies derived from different clones remained completelyunaffected by the same acid conditions.

FIG. 3 shows that the binding interaction of this D6B3 antibody was muchless sensitive to release at pH 5.0, with only 25 percent havingdissociated by 30 minutes in contrast to 80 percent at pH 4.5. Thekinetics of release for two monoclonal antibodies which did dissociateat pH 5.0, at 37° C. is shown in FIG. 4. The binding interaction betweenD5A7 and diphtheria toxin was even disrupted at pH levels as high as5.5. Thus a substantial fraction of diphtheria toxin was rapidlyrelinquished by these different antibodies at the precise pH andtemperature conditions found in endosomal vesicles and other acidiccompartments within cells (Geisow, J. L. and W. H. Evans, Exp. CellRes., 150:36-46 (1984)).

The pH-dependent break-up of antibody and toxin was shown to be basedupon conformational changes in the toxin. Thus, the t.sub. 1/2 ≈1-2 minfor the acid triggered dissociation of antibody and toxin is close tothe t.sub. 1/2 =30 sec for the pH-induced transition of free toxin(Blewitt, M. G., et al., Biochem., 24:5458-5464 (1985)). Moreover, theD6B3 antibody bound to formalin stabilized diphtheria toxoid at pH 7.0but did not release when the pH was reduced to pH 4.5. Apparently, thechemical crosslinking of toxoid prevented the pH-induced transitionwhich allows D6B3 to dissociate from native toxin.

EXAMPLE 2 Hybrid-Mediated Delivery of ¹²⁵ I-Diphtheria Toxin to Cells

Hybrid antibodies were formed with various anti-diphtheria toxinantibodies by linking them to anti-transferrin receptor monoclonalantibodies by a method previously described (Raso, F., et al., NATOAdvanced Studies Institute, 82:119-138 (1984)). The dual specificity andcell targeting capability of these hybrids was demonstrated using ¹²⁵I-diphtheria toxin (hereinafter ¹²⁵ I-DT). CEM cells derived from apatient with T-cell leukemia (Foley, G. E., et al., Cancer, 18:522-529(1965)), which have abundant transferrin receptor on their surface, wereused as a test line for anti-transferrin receptor/antidiphtheria toxinhybrids and two different routes of delivery were tested. The cells wereeither pre-treated with the hybrid and washed so that the empty toxinbinding sites of surface-bound hybrids could then capture subsequentlyadded ¹²⁵ I-DT; or hybrid plus ¹²⁵ I-DT were pre-complexed and then usedas a single agent for reaction with the cell surface transferrinreceptors.

CEM cells were incubated with the components designated in Table I for30 minutes at 0° and then washed with PBS to remove unbound hybrid. Theywere then exposed to ¹²⁵ I-DT for 30 minutes at 0°, washed with PBS andcounted to measure the amount bound to cells.

The results in Table I show that cells exposed to an anti-transferrinreceptor/anti-diphtheria toxin hybrid (7D3/D1F3) bound five times higherlevels of ¹²⁵ I-DT than untreated cells. This enhanced binding wasreceptor-specific since preoccupying the target epitope using excessunmodified 7D3 antibody blocked hybrid attachment and subsequent ¹²⁵I-DT binding (Table I). Hybrids formed with different anti-diphtheriatoxin monoclonal antibodies (D4B7 and D5E8) showed similar toxin bindingproperties (Table I).

                  TABLE I                                                         ______________________________________                                        Binding of .sup.125 I-DT to Hybrid-Coated CEM Cells                           Pretreatment         CPM Bound                                                ______________________________________                                        None                 888                                                      7D3/D1F3 Hybrid      4,381                                                    Excess 7D3 plus 7D3/D1F3 Hybrid                                                                    973                                                      None                 556                                                      7D3/D4B7 Hybrid      4,306                                                    7D3/D5E8 Hybrid      5,657                                                    ______________________________________                                    

CEM cells were treated for 1 hour at 0° C. with an equivalent amount of¹²⁵ I-DT either alone in PBS or pre-complexed at 22° for 15' to hybridat 10⁻⁸ M (Table II). Following treatment, the cells were washed withPBS and counted. Table II shows that significant delivery over the basalbinding levels was attained even though the concentration of complexused to treat these cells was relatively low (10⁻⁸ M).

                  TABLE II                                                        ______________________________________                                        Delivery of Hybrid-Complexed .sup.125 I-DT to CEM Cells                       Treatment             CPM Bound                                               ______________________________________                                        .sup.125 I-DT alone    1,649                                                  7D3/D4B7 Hybrid - .sup.125 I-DT complex                                                             13,116                                                  7D3/D5E8 Hybrid - .sup.125 I-DT complex                                                             15,297                                                  ______________________________________                                    

EXAMPLE 3 Plate Assay for Dual Specificity of HIV-Directed Hybrids

An anti-HIV monoclonal antibody was elicited using a synthetic envelopeprotein and used to form the HIV-specific hybrid (anti-HIV/D5E8) bycoupling it to an anti-diphtheria toxin antibody (D5E8) following amethod previously described (Raso, F., et al., NATO Advanced StudiesInstitute, 82:119-138 (1984)). A solid-phase radioimmunoassay wasdevised by adsorbing the envelope peptide antigen to the wells ofpolyvinyl microtitre plates. PBS and either antibody or hybrid at 6×10⁻⁹M was then added to the well for 2 hrs, and any unbound reagent waswashed off using PBS. The dual specificity of the hybrid wasdemonstrated after allowing it to bind to the coated plate via itsHIV-specific combining sites and then revealing its presence by binding¹²⁵ I-CRM107 to the free toxin-specific sites of the composite molecule.Table III shows that the anti-HIV/D5E8 hybrid bound ¹²⁵ I-CRM107 whileanti-HIV alone bound no toxin even though it was attached to the plateas evidenced by using an ¹²⁵ I-goat anti-mouse IgG probe.

                  TABLE III                                                       ______________________________________                                        Plate Assay to Demonstrate the Binding of                                     Anti-HIV Antibody and Hybrid                                                                  Amount Bound (CPM)                                                            .sup.125 I-CRM107                                                                      .sup.125 I-G/M                                       ______________________________________                                        PBS               307        253                                              anti-HIV          112        1,501                                            anti-HIV/D5E8 Hybrid                                                                            1,245      --                                               ______________________________________                                    

EXAMPLE 4 Hybrid-Mediated Cytotoxicity of a Mutated Form of DiphtheriaToxin

The availability of genetically or chemically altered diphtheria toxincogeners (e.g., CRM107) with no capacity for attaching to cells providesan added dimension to the hybrid delivery approach. The cell-bindingdefect which makes these analogs non-toxic to cells can be restored viathe hybrid carrier moiety so that its lethal action is aimed exclusivelyat the selected cell surface target.

Human mesothelioma cells (H-Meso) were used to test the effectiveness ofanti-transferrin receptor/anti-diphtheria toxin hybrids (7D3/D1F3 and7D3/D5E8) for restoring the full cytotoxic potential of CRM107. TheH-meso cells were incubated for 2 hours at 37° C. with 4×10⁻⁸ M CRM107alone; (4×10⁻⁸ M) CRM107 in combination with the hybrids 7D3/D5E8 or7D3/D1F3 at 1×10⁻⁸ M, or 4×10⁻⁸ M CRM107 in combination with the hybrids(1×10⁻⁸ M) plus excess anti-receptor antibody (7D3) (10⁻⁵ M). Cells werethen pulse labeled with ³ H-leucine for 30 min. H-meso cells in media towhich 10 mM NH₄ Cl was added were also incubated with the samecomponents.

The data in Table IV show that while CRM107 alone was incapable ofentering cells and inhibiting protein synthesis, it became a very potentand rapid-acting cytotoxin when used in combination with the hybridantibodies. This lethal action was dependent upon hybrid-mediateddelivery to transferrin receptors since little toxicity was obtainedwhen these sites were blocked by including an excess of freeanti-receptor antibody (7D3) during the 2 hour incubation time (TableIV).

The acid environment of intracellular compartments is essential forcytotoxicity since this induces the release of CRM107 from the antibodyand translocation into the cytosol where it inactivates elongationfactor 2. This condition was demonstrated by adding NH₄ Cl to the cells.This weak base, which is known to raise vesicle pH, greatly reduced theability of the hybrid-CRM107 combination to kill H-Meso cells (TableIV). The same experiments were carried out using the anti-HIV/D5E8hybrid (2×10⁻⁸ M) plus CRM107 (4×10⁻⁸ M) using HIV-infected 8E5 cells asthe target (Folks, T. M., et al., J. Exp. Med., 164:280-290 (1986)). Thesame acid-dependency was demonstrated (Table V).

                  TABLE IV                                                        ______________________________________                                        Hybrid-Mediated Cytotoxicity of CRM107 Tested on Human                        Mesothelioma Cells (2-hr Assay); Transferrin Receptor                         Specificity and Acid-Dependency                                                                .sup.3 H-Leucine                                                              Incorporation                                                                          Inhibition                                                           (CPM)    (Percent)                                           ______________________________________                                        H-Meso Cells       92,560     --                                              +CRM107            90,755      2                                              +7D3/D5E8 + CRM107  1,605     98                                              +7D3/D1F3 + CRM107  8,050     91                                              +excess 7D3 + 7D3/D5E8 +                                                                         52,325     43                                              CRM107                                                                        +excess 7D3 + 7D3/D1F3 +                                                                         53,960     42                                              CRM107                                                                        H-Meso Cells + 10 mM NH.sub.4 Cl                                                                 92,435     --                                              +CRM107            76,885     17                                              +7D3/D5E8 + CRM107 52,105     44                                              +7D3/D1F3 + CRM107 80,802     13                                              ______________________________________                                    

                  TABLE V                                                         ______________________________________                                        Acid-Dependency of Hybrid-Mediated Cytotoxicity of                            CRM107 on HIV-Positive 8E5 Cells                                                               .sup.3 H-Leucine                                                              Incorporation                                                                          Inhibition                                                           (CPM)    (percent)                                           ______________________________________                                        HIV-Positive + 8E5 cells alone                                                                   103,955    --                                              +CRM107            85,140     18                                              +NH.sub.4 Cl + CRM107                                                                            82,115     21                                              +anti-HIV/D5E8 + CRM107                                                                          21,820     79                                              +NH.sub.4 Cl + anti-HIV/D5E8 +                                                                   78,985     24                                              CRM107                                                                        ______________________________________                                    

The transferrin receptor directed hybrid-CRM107 complex was assayed onhuman colon adenocarcinoma cells to determine if the same high cytotoxicpotency found for the HoMeso and HIV-infected 8E5 cell lines extended toalternative malignant cell types. The combined action of CRM107 plushybrid at 10⁻⁸ M produced extensive cell kill within two hours and itspotency was comparable to 10⁻⁷ M native diphtheria toxin (Table VI).These results indicate that hybrid-delivery not only renders CRM107cytotoxic to cells but also suggests that its entry via the transferrinpathway is as efficient as diphtheria toxin uptake by its usualmechanism. Moreover, a transferrin/D5E8 conjugate was constructed toexamine if transferrin itself would mediate delivery of CRM107 intocells. In fact, this natural ligand coupled to the anti-diphtheria toxinmonoclonal antibody (D5E8) provided a similar level of toxicity as theanti-transferrin receptor (7D3) guided hybrid.

                  TABLE VI                                                        ______________________________________                                        Lethal Effects of Anti-Transferrin Receptor Directed Hybrid                   Plus CRM107 on Human Colon Adenocarcinoma Cells (2-hr.                        Assay)                                                                                         .sup.3 H-Leucine                                                              Incorporation                                                                          Inhibition                                                           (CPM)    (Percent)                                           ______________________________________                                        LS174T cells       54,070     --                                              +CRM107 (10.sup.-7 M)                                                                            48,355     11                                              +7D3/D5E8 (10.sup.-8 M) +                                                                        1,930      96                                              CRM107 (10.sup.-7 M)                                                          +Diphtheria Toxin (10.sup.-7 M)                                                                  1,785      97                                              +Diphtheria Toxin (10.sup.-8 M)                                                                  6,295      88                                              ______________________________________                                    

In addition to using the transferrin receptor as a target for hybriddelivery, the common acute lymphoblastic leukemia antigen (CALLA) wassimilarly tested as a site of entry into CALLA-bearing Nalm-1 leukemiacells (Raso, V., et al., Cancer Res., 42:457-464 (1982)). Ananti-CALLA/D5E8 hybrid was formed and examined for its ability to killthese cells in combination with CRM107 following the protocol set forthfor H-meso cells and anti-transferrin receptor/anti-diphtheria toxin.However, incubation was carried out for 6 hours at the same temperature(Table VII).

Good cell kill was achieved by targeting the hybrid-CRM107 to thisdistinct membrane site; however, the longer incubation time requiredsuggests that entry and/or release of toxin was slower than fortransferrin receptor directed agents.

                  TABLE VII                                                       ______________________________________                                        CALLA-Directed Cytotoxic Action of                                            Hybrid-CRM107 on Nalm 1 Cells (6 hr. Assay)                                                .sup.3 H-Leucine Incorporated                                                                Inhibition                                                     (CPM)          (Percent)                                         ______________________________________                                        Nalm-1 Cells   22,130           --                                            +anti-CALLA/D5E8                                                                             24,110           0                                             +CRM107        22,820           0                                             +anti-CALLA/D5E8 +                                                                            4,080           82                                            CRM107                                                                        ______________________________________                                    

EXAMPLE 5 Kinetics of Cytotoxicity in H-Meso Cells

One of the fundamental premises underlying the acid-triggered hybridcarrier concept predicts that this mode of delivery will not interferewith the normal mechanism of toxin action after specific targeting hasbeen achieved. A critical measure of toxin efficiency can be obtained bymonitoring the kinetics of inhibition of protein synthesis. Thisparameter accurately indicates how rapidly toxin gains access to itstarget in the cytosol (e.g., elongation factor 2) and was therefore usedto evaluate hybrid-delivered CRM107 (FIG. 5).

H-Meso cells were incubated at 37° C. for the designated intervals witheither 10⁻⁸ M diphtheria toxin, 10⁻⁸ M CRM107, or the anti-transferrinreceptor/anti-diphtheria toxin hybrid (7D3/D3E1)-CRM107 combination at10⁻⁸ M. The cells were then pulse labeled with ³ H-leucine for 30minutes to measure the extent incorporation into protein compared tountreated control cells. The time course of protein synthesis inhibitionas reflected by ³ H-leucine incorporation, for H-Meso cells incubatedwith 10⁻⁸ M diptheria toxin alone, 10⁻⁸ M CRM107 alone or with theanti-transferrin receptor/anti-diphtheria toxin hybrid (7D3/D3E1) plusCRM107 at 10⁻⁸ M was then measured.

FIG. 5 shows that both native toxin and the CRM107 hybrid combinationgave identical kinetics profiles which were characterized by a 30-40minute lag period followed by a rapid inactivation phase with t.sub. 1/2=24 minutes and t.sub. 1/2 =26 minutes respectively. Unbound CRM107alone at 10⁻⁸ M had no effect on the ability of the cells to synthesizeprotein. The fact that hybrid-delivered CRM107 killed cells as fast asnative diphtheria toxin suggests that its release from the antibodycombining site was unimpeded and that there was no interruption of thenormal course of events required for its lethal action.

Finally, a covalently-coupled anti-transferrin receptor-CRM107 conjugate(7D3-CRM107) was constructed by standard disulfide-linkage methods andits cytotoxic effect compared with the effect produced by the 7D3/D5E8hybrid plus CRM107. Transferrin receptor positive CEM cells wereincubated for 16 hours at 37° C. with the designated concentrations ofthe 7D3/D5E8 hybrid plus 10⁻⁷ M CRM107, the 7D3-CRM107 disulfide-linkedcovalent conjugate and native diphtheria toxin, CRM107 alone or 7D3/D5E8hybrid alone. The cells were then pulse labeled with ³ H-leucine for 30minutes and the amount of incorporation into protein was compared withuntreated control cells.

FIG. 6 shows the toxicity dose response curves of the hybrid, theconjugate and native diphtheria toxin. The conjugate, 7D3-CRM107 wascytotoxic to transferrin receptor positive cells, the kinetics of cellkilling was much slower than that found for hybrid-delivered CRM107. CEMcells are not particularly sensitive to diphtheria toxin as reflected inthe ID₅₀ =2×10⁻⁹ M obtained with native toxin. The transferrin-receptordirected 7D3-CRM107 conjugate was slightly more effective, giving anID₅₀ =1×10⁻⁹ M. In contrast, hybrid-delivered CRM107 (ID₅₀ =4×10⁻¹² M)was 250-fold more potent than the covalent conjugate, based upon theconcentration of hybrid added. Neither the 7D3/D5E8 hybrid alone norCRM107 alone had an effect upon the cells. These results indicate thatcovalent coupling can impede toxin action since the disulfide-linked7D3-CRM107 conjugate was slower acting and less potent than thecorresponding 7D3/D5E8 hybrid delivered CRM107.

FIG. 7 shows dose response curves for inhibition of protein synthesis inHIV-infected 8E5 cells after 16 hr exposure to CRM107 plus hybridsdirected against either HIV or transferrin receptors on the cellmembrane. The ID₅₀ for the anti-HIV/D5E8 hybrid plus CRM107 was 2×10⁻⁹ Mbut this reagent became 10-times more potent when nicked CRM107 (cleavedat a specific site using trypsin) was used (ID₅₀ =2×10⁻¹⁰ M). It isbelieved that proteolytic cleavage is a prerequisite for toxic activityand normally occurs at the cell surface or in subcellular compartments.This anti-HIV hybrid-mediated cytotoxicity was blocked by neutralizingintracellular compartments with NH₄ Cl (Table V) and the uninfectedcontrol cell line was not affected by hybrid-delivered CRM107.

Equivalents

Those skilled in the art will know, or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments of the invention as described herein. These and all otherequivalents are intended to be encompassed by the following claims.

I claim:
 1. A method for delivering a bioactive molecule having anantibody binding site that is sensitive to pH to a cellular targetwithin cells of a host, comprising:a) providing a first portion of ahybrid reagent which, upon administration to said cells at physiologicpH, binds to the surface of said cells with the subsequent pinching offof the surface of said cells to form endosomes having a second and lowerpH and containing said hybrid reagent whereby the hybrid reagent istransported to the interior of said cells by endocytosis, said firstportion comprising an antibody or antigen binding fragment thereof; and,b) screening antibodies or antigen binding fragments thereof which bindthe bioactive molecule to determine which of said antibodies or antigenbinding fragments bind the bioactive molecule at physiologic pH andrelease it at said second and lower pH found within the endosome; c)selecting an antibody or antigen binding fragment thereof to provide asecond portion of said hybrid reagent which binds said bioactivemolecule at physiologic pH and releases it at said second and lower pH;and d) constructing a hybrid reagent containing said first portion andsaid second portion; e) administering said hybrid reagent to said hostwhereby said constructed hybrid reagent binds to the surface of saidcells, is endocytosed into said cells and releases the bioactivemolecule to the cellular target with in said cells.
 2. A method of claim1 wherein said first pH is in the range of from about 6.5 to about 7.5.3. A method of claim 2 wherein said second and lower pH is in the rangeof from about 4.5 to about 5.5.
 4. A method of claim 1 wherein saidbioactive molecule is a toxin, an enzyme, a drug or a metal.
 5. A methodof claim 1 wherein said bioactive molecule is diphtheria toxin or acytotoxic mutant or cytotoxic fragment thereof.
 6. A method of claim 5wherein said first portion comprises an antibody or antigen bindingfragment thereof specific for transferrin receptor.
 7. A method of claim6 wherein said first pH is in the range of from about 6.5 to about 7.5and said second and lower pH is in the range of from about 4.5 to about5.5.
 8. A method of claim 1 wherein said hybrid reagent is combined witha pharmaceutically acceptable carrier.
 9. A method of claim 1 whereinsaid first portion binds to a cell surface receptor.
 10. A method ofclaim 9 wherein said cell surface receptor is a tumor-associatedreceptor.
 11. A method of claim 9 wherein said cell surface receptor isa viral-associated receptor.
 12. A method for delivering a bioactivemolecule having an antibody binding site that is sensitive to pH to acellular target within the cells of a host, comprising:a) providing afirst portion of a hybrid reagent which delivers and binds to thesurface of said cells at a first pH, with the subsequent pinching off ofthe surface of said cells to form endosomes having a second and lower pHand containing said hybrid reagent whereby the hybrid reagent istransported to the interior of said cells by endocytosis, said firstportion comprising an antibody or antigen binding fragment thereof; and,b) screening antibodies or antigen binding fragments thereof which bindthe bioactive molecule to determine which of said antibodies or antigenbinding fragments bind the bioactive molecule at said first pH andrelease it at said second and lower pH within the endosome; c) selectingan antibody or antigen binding fragment thereof to provide a secondportion of said hybrid reagent which binds said bioactive molecule atsaid first pH and releases it at said second and lower pH; and d)constructing a hybrid reagent containing said first portion and saidsecond portion; e) administering said hybrid reagent to said hostwhereby said constructed hybrid reagent with the antibody or antigenbinding fragment bound to the bioactive molecule binds to the surface ofsaid cells, is endocytosed into said cells and releases the bioactivemolecule to the cellular target with in said cells.